Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E.coli

Microb Cell Fact. 2011 Jan 7;10:1. doi: 10.1186/1475-2859-10-1.


Background: Disulfide bonds are one of the most common post-translational modifications found in proteins. The production of proteins that contain native disulfide bonds is challenging, especially on a large scale. Either the protein needs to be targeted to the endoplasmic reticulum in eukaryotes or to the prokaryotic periplasm. These compartments that are specialised for disulfide bond formation have an active catalyst for their formation, along with catalysts for isomerization to the native state. We have recently shown that it is possible to produce large amounts of prokaryotic disulfide bond containing proteins in the cytoplasm of wild-type bacteria such as E. coli by the introduction of catalysts for both of these processes.

Results: Here we show that the introduction of Erv1p, a sulfhydryl oxidase and a disulfide isomerase allows the efficient formation of natively folded eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli. The production of disulfide bonded proteins was also aided by the use of an appropriate fusion protein to keep the folding intermediates soluble and by choice of media. By combining the pre-expression of a sulfhydryl oxidase and a disulfide isomerase with these other factors, high level expression of even complex disulfide bonded eukaryotic proteins is possible

Conclusions: Our results show that the production of eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli is possible. The required exogenous components can be put onto a single plasmid vector allowing facile transfer between different prokaryotic strains. These results open up new avenues for the use of E. coli as a microbial cell factory.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cytoplasm / genetics
  • Cytoplasm / metabolism
  • Disulfides / metabolism*
  • Escherichia coli / cytology
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Gene Expression*
  • Genetic Engineering / methods*
  • Humans
  • Oxidoreductases / genetics
  • Oxidoreductases / metabolism*
  • Protein Disulfide-Isomerases / genetics
  • Protein Disulfide-Isomerases / metabolism
  • Protein Processing, Post-Translational
  • Protein Transport
  • Recombinant Proteins / genetics*
  • Recombinant Proteins / metabolism*


  • Disulfides
  • Recombinant Proteins
  • Oxidoreductases
  • sulfhydryl oxidase
  • Protein Disulfide-Isomerases